Biological systems use ligand-dependent proteins and nucleic acids as molecular switches to transduce inputs into appropriate cellular responses. For example, the activity of many enzymes is regulated by the presence of allosteric effectors. Artificial molecular switches are of particular interest (Guo et al. Science 288:2042–2045, 2000; Lin et al. J. Am. Chem. Soc. 125:612–613, 2003; Picard, Methods Enzymol. 327:385–401, 2000; Clackson, Curr. Opin. Chem. Biol. 1:210–218, 1997; Gossen et al. Science 268:1766–1769; each of which is incorporated herein by reference), because they enable biological functions to be controlled by small molecule inputs chosen by the researcher rather than by nature.
Protein-splicing elements, termed inteins, can mediate profound changes in the structure and function of proteins. Inteins are analogous to the introns found in polynucleotides. During intein-mediated protein splicing, the intein is present in the mature mRNA and translated to form the precursor protein. Inteins catalyze both their own excision from within a polypeptide chain and the ligation of the flanking external sequences (exteins) resulting in the formation of the mature protein from the exteins, and the free intein. No natural inteins, however, have been shown to be regulated by small molecules. Extein function is typically disrupted by the presence of an intein but restored after protein splicing. Many inteins can splice in foreign extein environments. Therefore, inteins are powerful starting points for the creation of artificial molecular switches.
Muir and coworkers (Mootz et al. J. Am. Chem. Soc. 124:9044–45, 2002; Mootz et al. J. Am. Chem. Soc. 125:10561–69; each of which is incorporated herein by reference) recently described the fusion of the N- and C-terminal halves of the Saccharomyces cerevisiae VMA intein with the FKBP and FRB proteins, creating rapamycin-dependent splicing in trans. Modulation of protein function, however, has not yet been reported using the resulting split inteins, and their application may be limited by the instability of partially folded, split proteins.
A ligand-dependent intein on a single polypeptide chain that can be used to modulate the function of a target protein has yet to be generated. Such an intein could preferably be used in a variety of target proteins, thereby making such an intein very useful in elucidating the functions of various target proteins in vivo.